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United States Patent | 5,643,746 |
Polakowska ,   et al. | July 1, 1997 |
The present invention relates to promoter elements from human type I transglutaminase (TGase I) genes for controlled gene expression of both human TGase I and heterologous proteins. These promoter elements permit tissue-specific expression of genes, e.g. for use in human gene therapy and for testing pharmaceutical agents with artificial skin. Additionally, the subject promoter elements can provide differential regulation under physiological conditions or in the presence of exogenously added factors including calcium and retinoic acid.
Inventors: | Polakowska; Renata Regina (Pittsford, NY); Goldsmith; Lowell Alan (Pittsford, NY) |
Assignee: | Research Corporation Technologies, Inc. (Tucson, AZ) |
Appl. No.: | 216219 |
Filed: | March 21, 1994 |
Current U.S. Class: | 435/69.1; 435/320.1; 435/366; 435/371; 536/23.1 |
Intern'l Class: | C12N 015/63; C12N 015/79; C12N 015/09 |
Field of Search: | 536/23.1 435/320.1,240.2,172.3 935/22,70 |
4888291 | Dec., 1989 | Barrandon et al. | 435/240. |
Floyd, et al. (1989) Mol. Cell. Biol. 9:4846-4851, Regulation of Type I (Epidermal) Transglutaminase mRNA Levels During Squamous Differentiation: Down Regulation by Retinoids. Gentile, et al. (1989) J. Cell. Biol. 109:198A, Isolation and Characterization of cDNA and Genomic Clones of Human Endothelial Cell Tranglutaminase. Goldsmith, et al. (1991) J. Invest. Dermatol. 97: 156-158, Inhibition of Human Epidermal Transglutaminases In Vitro and In Vitro by Tyrosinamidomethyl Dihydrohaloisoxazoles. Haake, et al. (1991) J. Invest. Dermatol. 96:71-77, Physiologic Distribution and Differentiation of Melanocytes in Human Fetal and Neonatal Skin Equivalents. Ichinose, et al. (1988) Proc. Natl. Acad. Sci. USA 85:5829-5833,Characterization of the Gene for a Subunit of Human Factor XIII (plasma transglutaminase), a Blood Coagulation Factor. Jetten, A.M. (1991) International Publication No. WO91/06553; PCT Appln. No. PCT/US90/067075, Type I Transglutaminase DNA, published May 16, 1991, claiming priority of U.S. Serial No. 425887, filed Oct. 24, 1989. Kim, et al. (1991) J. Biol. Chem. 226:536-539,The Complete Amino Acid Sequence of the Human Transglutaminase K Enzyme Deduced from the Nucleic Acid Sequences of cDNA Clones. Michel, et al. (1989) FEBS Lett. 258:35-38, Retinoic Acid Controls Expression of Epidermal Translutaminase at the Pre-translational Level. Phillips, et al. (1990) Proc. Natl. Acad. Sci. USA 87:9333-9337, Primary Structure of Keratinocyte Transglutaminase. Polakowska, et al. (1991) J. Invest. Dermatol. 96:285-288, Isolation of cDNA cDNA for Human Epidermal Type I Transglutaminase. Polakowska, et al. (1990) J. Invest. Dermatol. 94:567,Cloning of Human Epidermal type I Transgutaminase (TGase). Schroeder, et al. (1990) Clin. Res. 38:962A, Isolation of a cDNA for Human Epidermal Transglutaminase (Type I). Yamanishi, et al. (1991) Biochem. Biophys. Res. Comm. 175: 906-913, Molecular Cloning of Human Epidermal Transglutaminase cDNA from Keratinocytes in Culture. Wolff et al. (1990) Science 247:1465-1468, Direct Gene Transfer into Mouse Muscle in Vivo. Morgan et al. (1990) Science 247:1465-1468, Direct Gene Transfer into Mouse Muscle in Vivo. St. Louis et al. (1988) Proc. Natl. Acad. Sci. 85:3150-3154 An Alternative Approach to Somatic Cell Gene Therapy. Fenjves et al. (1989) Proc. Natl. Acad. Sci. 86:8803-8807, Systemic Distribution of Apolipoprotein E Secreted by Grafts of Epidermal Keratinocytes: Implications for Epidermal Function. Fenjves et al. (1990) DNA Damage and Repair in Human Tissues, Sutherland et al., eds., Plenum Press, New York pp 215-223. Jensen et al. (1991) J. Cell Sci. 100:255-259, Tissue Culture of Human Epidermal Keratinocytes:a Differentiating Model System for Gene Testing and Somatic Gene Therapy. Miller (1990) Blood 76:271-278, Progress Toward Human Gene Therapy. International search Report PCT/US 93/00537. Phillips et al. (1992) J. Biol. Chem. 267:2282-2286, Genomic Structure of Keratinocyte Transglutaminase. Kim et al. (1992) J. Biol. Chem. 267:7710-7717, Structure and Organization of the Human Transglutaminase 1 Gene. Yamanashi et al. (1992) J. Biol. Chem. 267:17858-17863, Structure of the Gene for Human Transglutaminase 1. |
TABLE I ______________________________________ Selected Features of the Human TGase I Promoter Region Represented in SEQ ID NO: 1 Feature Nucleotide ______________________________________ TATA box 901-905 Exon I 936-1021 Intron 1 1022-1955 Exon II 1756-end.sup.a BamHI sites 485 and 1088 HindIII sites 616 and 1569 NlaIII sites 1563 and 1757.sup.b AUG initiation codon 1758 ______________________________________ .sup.a The full sequence of exon II is not provided. .sup.b Only the pertinent NlaIII sites are identified.
TABLE 2 ______________________________________ CAT Assay for Regulatory Elements in the Human TGase I Gene Promoter Region Plasmid.sup.a pSV2 pBL3 B H N 194+ None ______________________________________ Control.sup.b 91.9 0.84 4.59 0.11 18.1 8.61 0.09 Hi Ca.sup.2+ 86.5 0.37 8.37 0.11 8.12 6.25 0.10 Hi Ca.sup.2+ + 80.1 0.69 4.47 0.10 19.4 7.63 0.09 RA ______________________________________ .sup.a The indicated plasmids were transiently transfected into primary neonatal keratinocytes and assayed for CAT activity. Activity is expresse as percent acetylated. The plasmids are as follows: pSV2, pSV2cat (positive control); pBL3, pBLCAT3 (negative control); B, BamHI fragment i pBLCAT3; H, HindIII fragment in pBLCAT3; N, NlaIII194 fragment in pBLCAT3 194+, HindIIINlaIII-194 fragment in pBLCAT3; None, no DNA. .sup.b Control is low calcium (0.07 mM Ca.sup.2+); Hi Ca.sup.2+ is 1.2 m Ca.sup.2+ ; Hi Ca.sup.2+ + RA is 1.2 mM Ca.sup.2+ and 10.sup.-6 M retinoic acid.
__________________________________________________________________________ SEQUENCE LISTING (1) GENERAL INFORMATION: (iii) NUMBER OF SEQUENCES: 1 (2) INFORMATION FOR SEQ ID NO:1: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 2003 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: both (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1: GAGCTCGAGGATCGCCTTGGTGCCATCCTCAGTGTTTCCCCAGTGTCGGGGTGGAGTAAG60 GATGCACACAGCACAGGTGCCTGAGCCGGTTGGTCTGGATTCCCAGGAAGAATTCCAGGT120 TGGAGAAAAGGGAATCCTCTAGTCCAGCTGAGCAGAGACTCCTCTCAGTGAGAGAAAGGT180 ACTCTGTACCCCTGGAAGGGACTCAGTTCCCACCCAAGCCTGAGTGGAAAGGCCTAAACA240 TCCCCTAACCCCCGAGGCTACAGCGGGGGTGGGGGACGTGAAATGAGATTGCTCCTACTC300 TGATCTCCCTAATCCCAAACTTGAGGGCAGCTCACTCATGCCTGGGGCTGTAGAGCAGCT360 GAGAAAGAAGGGACAGACTTGGGGGTGGAGGGAGTCAGAATATCTGGTAGAGCCAGCAGG420 TCAGGGGTTAGCTGGTGGAGCCAGTCTGAGGGCCTGGCTGCTGATGTCACCAGTCTGCAA480 CCTGGGATCCCAGGACCTCCCTGGGCAGGATGAGTTCCAGGACCAGGCCCCTGGGCCAAT540 TTCATAGGGCTGAGCCTGGCTTGGGCTGCACAGAACTCGGCAGCAGGAGCCTGTGACAGC600 AGAGGTAGGCAGCCTAAGCTTGGGACCAGAAGGTCGGCCAGACAGGGCTGTGGGTGGAAG660 GGCCTGCCTGCCCCACTGCCCTTGCAGCTTCTTCATCCGGGAGAAGGGGCTCCTCACATG720 CCCAGTCTGGTAGGAATCAGCCTGGTGCCAGGGGCCATCACAGCGGTGGCTCCCACCAAA780 GCCCAGCCTAAGCCCCCAGACCTCACCCCTGCTCCCTCCCTAGCATCTTCTCCCCATTTC840 CCGCCCAGAGGCCTGGCCTCTTCTCTCCGCCCCCTACAGCAGTTTGGCCCCTCCCTCCCA900 CATAAGTCACTTACCAGGTCTGTCCCTGCGGCATCCAGTCTGTGGGTCCTGTCCCATCCA960 TCCTGACCTGTTCCATCTCAGCCCCAGGACTCAGTACTGCGGTTGCCAACACTGCTGCCA1020 GGTGAGGGGCTCCCACGGGTACTGTGGTGCCGAGTCCAGGCGGCCCACACTATCAGAGGC1080 CGTGCCTGGATCCAGCAAGGTGGGGTGTGGGCCAGCTGTGTACCTGTCAGCCCCAGCTAG1140 GCTGTTCCCAACACCAAGACTCTGCTTTCCCTGTGCACAGGCTCCGGGCACCTGCCATGC1200 CCTACCCCTCCTGGCAGCCCCAAGTGGGGTCTTCCCTGACTTGGGAATGCCAAGGACCAC1260 AGGCCCCGGGGTCACTTGTCTGTCTTGTGAGGAACCTTGAGTTGGGGGATTTCTGCTAAG1320 AAATGAGTTCTAGAAGCTGTCAGTGTTGTGCACCTCTAGACTGCAGAGCTAGCAGGTGGA1380 CGGACCAGGCCCAGGGATGCTGGAGCACTCTGATGTGTGTGCAGCTGGGTCTTGAGGCTG1440 GGACAAGTGTCCATGCAGGGAACTATGTGGATTTCCTGGGATGGATCGTTGAGTGGGTTT1500 CTTCATTGGGCAGTTTTTCGGATGTTGTTTGGTGGGGGTGAGGGGAAGGCTTTTCTCTGC1560 AGCATGAGAAGCTTCTCTGGGTGAGTCTGAATGGTCTTCGCGGAAGGTCTCTGGATGTGT1620 CTGGAGAATCTCTGGGCCAAGGTGGGATTGTTTCGGTCATCGGGTGGGACTGAATCAGCT1680 GTCTGGATGGAGGGTTTCTGGGTCAACTGGCTGGGACTACCTGGGTTAAGGAGCCACCCT1740 GCCTCTTCCTAACAGGCATGATGGATGGGCCACGTTCCGATGTGGGCCGTTGGGGTGGCA1800 ACCCCTTGCAGCCCCCTACCACGCCATCTCCAGAGCCAGAGCCAGAGCCAGACGGACGCT1860 CTCGCAGAGGAGGAGGCCGTTCCTTCTGGGCTCGCTGCTGTGGCTGCTGTTCATGCCGAA1920 ATGCGGCAGATGACGACTGGGGACCTGAACCCTCTGACTCCAGGGGTCGAGGGTCCAGCT1980 CTGGCACTCGAAGACCTGGCTCC2003 __________________________________________________________________________